Imidazolinone and sulfonylurea herbicides are widely used in modem agriculture due to their effectiveness at very low application rates and relative non-toxicity in animals. Imidazolinone and sulfonylurea herbicides inhibit the activity of acetohydroxyacid synthase (AHAS), or acetolactate synthase (ALS) (E.C.4.1.3.18), the key enzyme in the biosynthesis of branch chain amino acids such as valine, leucine and isoleucine (Shaner et al. 1984 Plant Physiol. 76:545-546). By inhibiting AHAS activity, this class of herbicides prevents further growth and development of susceptible plants including many weed species. Several examples of commercially available imidazolinone herbicides are PURSUIJTS (imazethapyr), SCEPTER® (imazaquin) and ARSENAL® (imazapyr). Examples of sulfonylurea herbicides are chlorsulfiuron, metsulfuron methyl, sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl, nicosulfuron, ethametsulfuron methyl, rimsulfuron, triflusulfuron methyl, triasulfuron, primisulfuron methyl, cinosulffiron, amidosulfuron, fluzasulfuron, imazosulfuron, pyrazosulfiron ethyl and halosulfuron.
Due to their high effectiveness and low-toxicity, imidazolinone herbicides are favored for application by spraying over the top of a wide area of vegetation. The ability to spray an herbicide over the top of a wide range of vegetation decreases the costs associated with plantation establishment and maintenance and decreases the need for site preparation prior to use of such chemicals. Spraying over the top of a desired tolerant species also results in the ability to achieve maximum yield potential of the desired species due to the absence of competitive species. However, the ability to use such spray-over techniques is dependent upon the presence of imidazolinone resistant species of the desired vegetation in the spray over area.
Among the major agricultural crops, some leguminous species such as soybean are naturally resistant to imidazolinone herbicides due to their ability to rapidly metabolize the herbicide compounds (Shaner and Robinson 1985 Weed Sci. 33:469-471). Other crops such as corn (Newhouse et al. 1992 Plant Physiol. 100:882-886) and rice (Barrette et al. 1989 Crop Safeners for Herbicides, Academic Press New York, pp. 195-220) are somewhat susceptible to imidazolinone herbicides. The differential sensitivity to the imidazolinone herbicides is dependent on the chemical nature of the particular herbicide and differential metabolism of the compound from a toxic to a non-toxic form in each plant (Shaner et al. 1984 Plant Physiol. 76:545-546; Brown et al. 1987 Petic. Biochm. Physiol. 27:24-29). Other plant physiological differences such as absorption and translocation also play an important role in sensitivity (Shaner and Robinson 1985 Weed Sci. 33:469-471).
Computer-based modeling of the three dimensional conformation of the AHAS-inhibitor complex predicts several amino acids in the proposed inhibitor binding pocket as sites where induced mutations would likely confer selective resistance to imidazolinones (Ott et al. 1996 J. Mol. Biol. 263:359-368). Lentil plants produced with these rationally designed mutations in the proposed binding sites of the AHAS enzyme have in fact exhibited specific resistance to a single class of herbicides (Ott et al. 1996 J. Mol. Biol. 263:359-368). Other mutations in the AHAS gene have been linked to resistance to the imidazolinone herbicides in canola (Swanson et al. 1989 Theor. Appl. Genet. 78:525-530) and com (Newhouse et al. 1991 Theor. Appl. Genet. 83:65-70).
Studies of the ALS gene in other crop plants have also resulted in sulfonylurea and imidazolinone resistance in those plants. In one report, use of a mutant ALS gene from Arabidopsis coupled with selection on sulfonylurea herbicide resulted in the production of resistant transgenic rice plants (Li et al. 1992 Plant Cell Rep. 12:250-255). An increase in in vitro resistance to chlorsulfuron of similar magnitude (200-fold) was demonstrated in transgenic rice containing a 35S/ALS transgene (Li et al. 1992 Plant Cell Rep. 12:250-255), and imidazolinone-resistant growth of transgenic tobacco was 100-fold greater than non-transformed control plants (Sathasivan et al. 1991 Plant Physiol. 97:1044-1050).
Expression of the introduced AHAS or ALS gene at different magnitudes has also been achieved by manipulating several aspects of the transformation including the use of different promoters and screening larger populations of transformants (Odell et al. 1990 Plant Physiol. 94:1647-1654; Sathasivan et al. 1991 Plant Physiol. 97:1044-1050; Li et al. 1992 Plant Cell Rep. 12:250-255). Studies showed that replacing the Arabidopsis ALS promoter with the CaMV35S promoter resulted in 40-fold differences in in vitro resistance to chlorsulfuiron (Li et al. 1992 Plant Cell Rep. 12:250-255). In tobacco, the increase in resistance to imazethapyr in individual calli transformed with a mutant ALS gene from Arabidopsis ranged from 10- to 1000-fold, most likely reflecting the differences in gene copy numbers or in chromosomal positions of the transgenes (Sathasivan et al. 1991 Plant Physiol. 97:1044-1050).
Plant resistance to imidazolinone has also been reported in a number of patents. U.S. Pat. No. 4,761,373 generally describes the use of an altered AHAS gene to elicit herbicide resistance in plants, and specifically discloses certain imidazolinone resistant corn lines. U.S. Pat. No. 5,013,659 discloses plants exhibiting herbicide resistance possessing mutations in at least one amino acid in one or more conserved regions. The mutations described therein encode either cross-resistance for imidazolinones and sulfonylureas or sulfonylurea-specific resistance, but imidazolinone-specific resistance is not described. Additionally, U.S. Pat. No. 5,731,180 and U.S. Pat. No. 5,767,361 discuss an isolated gene having a single amino acid substitution in a wild-type monocot AHAS amino acid sequence that results in imidazolinone-specific resistance.
However, to date, the prior art has not described an imidazolinone resistant pulse crop such as lentil. Pulses are the seeds of legumes that are used as food, including pea, bean, lentil, chickpea and fababean. Pulse crops, provide about 10% of the total dietary protein of the world. Lentil was one of the earliest cultivated crops in the world with archeological evidence from the early Stone Age. Lentil remains an important source of dietary protein in India, Southwest Asia and the Mediterranean, and Canadian lentil production is primarily directed toward export to these regions. While lentil is grown mainly for the seed to be harvested as a food export, the straw can also be used as a high quality animal feed or as a source of organic material for soil improvement. Cultivated varieties of lentil (Lens culinaris) are believed to descend from Lens orientalis, the only wild-type species able to naturally cross with Lens culinaris and produce fully fertile progeny.
A major challenge in lentil production is weed control. Lentil seedlings are short and slow-growing in relation to many weed species and therefore compete very poorly. Effective chemical weed control is necessary for commercial viability. The ability to spray over an herbicide that kills a broad spectrum of broadleaf weeds, either as a pre-emergent spray or as a post-emergent spray, would be beneficial to lentil production. Even more advantageous would be an herbicide that also controls a broad spectrum of grassy weeds and volunteer cereals that could be applied over a broad area of lentil crops.
Therefore, what are needed in the art are lentil plants having increased resistance to herbicides such as imidazolinone and methods for controlling weed growth in the vicinity of lentil plants. Such compositions and methods would allow for the use of spray over techniques when applying herbicides to areas containing lentil plants.